In higher eukaryotes, the sarco-endoplasmic reticulum (ER) Ca(2+)-ATPase (SERCA) is characterized for its high sensitivity to low concentrations of thapsigargin (TG), a very specific inhibitor. In contrast, SERCA-like enzymes with different sensitivities to TG have been reported in trypanosomatids. Here, we characterized a SERCA-like enzyme from Trypanosoma evansi and evaluated its interaction with TG. Confocal fluorescence microscopy using BODIPY FL TG and specific anti-SERCA antibodies localized the T. evansi SERCA-like enzyme in the ER and confirmed its direct interaction with TG. Moreover, the use of either 1 μM TG or 25 μM 2',5'-di (tert-butyl)-1,4-benzohydroquinone prevented the reuptake of Ca(2+) and consequently produced a small increase in the parasite cytosolic calcium concentration in a calcium-free medium, which was released from the ER pool. A 3035 bp-sequence coding for a protein with an estimated molecular mass of 110.2 kDa was cloned from T. evansi. The corresponding gene product contained all the invariant residues and conserved motifs found in other P-type ATPases but lacked the calmodulin binding site. Modeling of the three-dimensional structure of the parasite enzyme revealed that the amino acid changes found in the TG-SERCA binding pocket do not compromise the interaction between the enzyme and the inhibitor. Therefore, we concluded that T. evansi possesses a SERCA-like protein that is inhibited by TG.
Keywords: SERCA; Thapsigargin; Trypanosoma evansi.
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